Lactase or β-galactosidase (E.C: 3.2.1.23) is an enzyme which catalyses the hydrolysis of lactose (a disaccharide) into its component monosaccharides glucose and galactose. Lactose is present in dairy products and more specifically in milk, skimmed milk, cream, yoghurt, ice-cream and other milk products.
In juveniles and people that are lactose tolerant, lactose is hydrolyzed into galactose and glucose by the “lactase-phlorizin hydrolase” (LPH) in the small intestinal brush border of the jejunum. LPH is encoded by a single lactase gene LCT in humans, and it is found that the ability to digest lactose in adulthood is due to cis-acting mutations in gene expression and inherited in a dominant manner. Lactose, in contrast to the mono-sugars (i.e., glucose and galactose), is poorly absorbed in the small intestine. People that do not carry mutations in the LCT gene will not digest and absorb lactose, and malabsorbed lactose will osmotically attract fluid into the bowel lumen which will lead to a loose stool. Additionally, lactose is a substrate for intestinal bacteria in the colon that metabolize it, producing volatile fatty acids and gases such as carbon dioxide, hydrogen and methane, leading to flatulence and cramping. Current estimate is that only 25-30% of the world population carries the mutation in the LCT gene and is also at adulthood able to digest lactose in the small intestine. The rest of the world population has varying difficulty with lactose digestion and this often leads to reduced intake of dairy products. Such individuals are called “lactose intolerant”. Since dairy products are an important component of a healthy diet, due to the presence of several vitamins, proteins and minerals, this is an unwanted situation.
Lactases have been described for and isolated from a large variety or organisms, including micro-organisms. Lactase is often an intracellular component of micro-organisms like Kluyveromyces and Bacillus. Kluyveromyces and especially K. fragilis, K. marxianus and K. lactis, and other yeasts such as those of the genera Candida, Torula and Torulopsis are a common source of yeast lactases, whereas B. coagulans or B circulans are well known sources for bacterial lactases. Several commercial lactase preparations, derived from these organisms are available such as Maxilact (from K. lactis, produced by DSM, Delft, the Netherlands). All these lactases are so called neutral lactases since they have a pH optimum between pH=6 and pH=8. Several organisms such as Aspergillus niger and Aspergillus oryzae can produce extracellular lactase, and U.S. Pat. No. 5,736,374 describes an example of such lactase, produced by Aspergillus oryzae. The enzymatic properties of lactases like pH- and temperature optimum vary between species. In general, however, lactases that are excreted show a lower pH-optimum of pH=3.5 to pH=5.0 (acid lactases); intracellular lactases usually show a higher pH optimum in the region of pH=6.0 to pH=7.5 for neutral lactases, but exceptions on these general rules occur.
Currently, lactase is formulated and commercially sold as a liquid. Two factors are of great importance when producing a liquid formulation, namely microbial and enzymatic stability. With microbial stability is meant that the liquid formulation stays free of microorganisms, or at least that growth is prevented or slowed down.
Several liquid lactase products are currently on the market. Examples of these (neutral lactases only) are Lactozym Pure 6500 L, sold by Novozymes; Ha-Lactase 5200, sold by Chr. Hansen; GODO YNL-2, sold by DuPont-Danisco; and Maxilact LGX 5000 and LGi, both sold by DSM Food Specialties. All the above products, and indeed all liquid lactase formulations presently on the market contain approximately 50% glycerol. It has been found that glycerol both affords good microbial and enzymatic stabilization. The amount of glycerol is such that the water activity (Aw) is at most 0.82. This prevents microbiological growth. In addition, the glycerol also affords enzymatic stability. A typical strength of a commercial lactase solution is about 1000, 2000 or 5000 NLU/g. The term NLU/g refers to the amount of lactase activity per gram end product.
An important industrial application of lactase is in the production of lactose-hydrolyzed milk products for lactose intolerant individuals. Such hydrolysed milk products include pasteurized milk, UHT-milk, milk reconstituted from all or part of its original constituents with or without intermediate processing steps such as protein hydrolysis, and infant formula.
A particularly interesting application of lactase is infant, follow-on or toddler formula. Infant formula is a manufactured food designed and marketed for feeding to babies and infants under 12 months of age, usually prepared for bottle-feeding or cup-feeding from powder (mixed with water) or liquid (with or without additional water). The U.S. Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formula as “a food which purports to be or is represented for special dietary use solely as a food for infants by reason of its simulation of human milk or its suitability as a complete or partial substitute for human milk”. The most commonly used infant formulas contain purified cow's milk whey and casein as a protein source, a blend of vegetable oils as a fat source, lactose as a carbohydrate source, a vitamin-mineral mix, and other ingredients depending on the manufacturer.
A problem with currently available lactase formulations is that in many countries the addition or carry-over of glycerol to infant, follow-on or toddler formula is prohibited by law. This makes the present lactase formulations, which all contain glycerol, unsuitable to produce infant, follow-on or toddler formula. Therefore, there is a need for stable liquid lactase formulations which are suitable for infant, follow-on or toddler formula and substantially free of glycerol.